Nucleic acid hybridizations are now commonly used in genetic research, biomedical research and clinical diagnostics. In the basic nucleic acid hybridization assay, single-stranded analyte nucleic acid (either DNA or RNA) is hybridized to a labeled nucleic acid probe, and resulting labeled duplexes are detected. Both radioactive and non-radioactive labels have been used.
Variations of this basic scheme have been developed to facilitate separation of the duplexes to be detected from extraneous materials and/or to amplify the signal that is detected.
Copending commonly owned U.S. Ser. No. 807,624, filed 11 Dec. 1985, described a solution-phase nucleic acid hybridization assay in which the analyte nucleic acid is hybridized to a labeling probe set and to a capturing probe set. The probe-analyte complex is coupled by hybridization with a solid-supported capture probe that is complementary to the capture probe set. This permits the analyte nucleic acid to be removed from solution as a solid phase complex. Having the analyte in the form of a solid phase complex facilitates subsequent separation steps in the assay. The labeling probe set is complementary to a labeled probe that is bound through hybridization to the solid phase/analyte complex.
PCT Application 84/03520 and EPA 124221 described a DNA hybridization assay in which analyte is annealed to a single-stranded DNA probe having a tail that is complementary to an enzyme-labeled oligonucleotide, and (2) the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide. The Enzo Biochem "Bio-Bridge" labeling system appears to be similar to the system described in these two patent applications. The "Bio-Bridge" system uses terminal deoxynucleotide transferase to add unmodified 3'-polyT-tails to a DNA probe. The polyT-tailed probe is hybridized to the target DNA sequence and then to a biotin-modified polyA.
EPA 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly-dT tail, an amplifier strand that has a sequence, e.g., a poly-dA sequence, that hybridizes to the tail of the probe and is capable of binding a plurality of labeled strands.
Collins et al, U.S. Pat. No. 4,818,680, disclosed a polynucleotide displacement assay in which the target DNA sequence displaces a labeled signal strand from a partially double-stranded probe. The displaced signal strand is captured by hybridization to a capture probe, and the amount of labeled signal strand left after washing is quantified.
Vary, U.S. Pat. No. 4,795,701, disclosed another polynucleotide displacement assay, in which the signal strand is preferably RNA, thus making the probe reagent a DNA/RNA probe/signal strand heteroduplex. The amount of signal strand displaced is quantified by digesting the displaced strand to individual nucleotides, converting the ADP so produced to ATP, and assaying the ATP by its reaction with luciferase. The drawbacks to this method are that it depends upon complete digestion of only the displaced signal strands, it is subject to high background levels from ATP naturally present in the sample, and that the signal will vary with the adenosine content of the signal strand.
The main problem with these prior hybridization assays is that they lack sufficient specificity and/or signal to be useful for detecting very low levels of analyte. A primary object of the present invention is to provide amplification for use in nucleic acid hybridizations that provides a high reproducible gain in signal, a high reproducible signal-to-noise ratio and low nonspecific binding, that is itself reproducible, and that is capable of combining specifically with a "universal" signal moiety and an analyte at low concentrations to form a stable complex.
An improvement in DNA amplification, the polymerase chain reaction (PCR) technique, was disclosed by Mullis in U.S. Pat. Nos. 4,683,195 (Mullis et al) and 4,683,202, incorporated herein by reference. In the PCR technique, short oligonucleotide primers are prepared which match opposite ends of a desired sequence. The sequence between the primers need not be known. A sample of DNA (or RNA) is extracted and denatured (preferably by heat). Then, oligonucleotide primers are added in molar excess, along with dNTPs and a polymerase (preferably Taq polymerase, which is stable to heat). The DNA is replicated, then again denatured. This results in two "long products," which begin with the respective primers, and the two original strands (per duplex DNA molecule). The reaction mixture is then returned to polymerizing conditions (e.g., by lowering the temperature, inactivating a denaturing agent, or adding more polymerase), and a second cycle initiated. The second cycle provides the two original strands, the two long products from cycle 1, two new long products (replicated from the original strands), and two "short products" replicated from the long products. The short products have the sequence of the target sequence (sense or antisense) with a primer at each end. On each additional cycle, an additional two long products are produced, and a number of short products equal to the number of long and short products remaining at the end of the previous cycle. Thus, the number of short products grows exponentially with each cycle. This amplification of a specific analyte sequence allows the detection of extremely small quantities of DNA.
The recent advent of PCR technology has enabled the detection of specific DNA sequences present in extremely minute (&lt;1 fg) quantities. However, in order to obtain accurate results near the detection limit, great care must be exercised to avoid contamination with foreign DNA. It is possible to amplify DNA present on the glassware or in the reagents rather than the DNA originating in the sample, thus producing erroneous results.